A fluorescent dye in the second near-infrared region, a fluorescent probe, and a preparation method and application thereof

Abstract

The main purpose of the present invention is to provide a near-infrared second region fluorescent dye, fluorescent probe and its preparation method and application. A near-infrared second region fluorescent dye, its general structural formula is as follows: wherein X is one of F, Cl, Br, and I.

Description

Technical Field

[0001] The present application relates to the field of compounds, and in particular to a near-infrared second region fluorescent dye, a fluorescent probe, and a preparation method and application thereof. Background Art

[0002] Fluorescence imaging in the second near-infrared window (NIR-II, 900-1700nm) is an emerging in vivo imaging technique for animals. It provides improved imaging depths up to several centimeters and higher imaging frame rates, which shows great potential in in vivo imaging. So far, various fluorophores have been reported for near-infrared second window fluorescence imaging. However, most of the current near-infrared second-region fluorescent dyes lack modifiable sites, so they are difficult to modify or drug trace.

[0003] To this end, the present invention is proposed. Summary of the invention

[0004] The main purpose of the present invention is to provide a fluorescent dye in the near-infrared region II, a fluorescent probe, and a preparation method and application thereof. The fluorescent dye has a carboxylic acid conjugated modification site and good chemical stability. The emission wavelength reaches the near-infrared region II and can be excreted from the body through the hepatobiliary metabolic pathway.

[0005] In order to achieve the above objectives, the present invention provides the following technical solutions.

[0006] The first aspect of the present invention provides a fluorescent dye in the second near-infrared region, the general structural formula of which is as follows:

[0007]

[0008] Wherein, X is one of F, Cl, Br and I, and preferably X is Br.

[0009] The fluorescent dye may be a mixture of multiple isomers or may be various isomers.

[0010] The present invention also synthesizes the trans isomer of the above fluorescent dye, the chemical name of which is:

[0011] 1-(5-Carboxypentyl)-3,3-dimethyl-2-((E)-2-(6-((E)-2-(2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-9-yl)vinyl)-2,3-dihydro-1H-xanthene-4-yl)vinyl)-3H-indole-1-bromide, referred to as VIPI.

[0012] The second aspect of the present invention provides a synthetic route of the above fluorescent dye:

[0013]

[0014] Wherein, Y is one of F, Cl, Br and I, with Br being preferred.

[0015] Compound 1 is mixed with compound 2, and reacted in the presence of a base, a catalyst and an organic solvent to obtain compound 3. Compound 3 and compound 4 are subjected to a condensation reaction in an acid and a salt to obtain the compound of formula I shown.

[0016] By adjusting the type, amount / ratio, reaction temperature, reaction time and other parameters of each reactant, a high-purity product can be obtained or the yield can be improved, as shown in the following preferred embodiments.

[0017] Preferably, the base is one of triethanolamine and triethylamine, preferably triethylamine.

[0018] Preferably, the catalyst is palladium acetate.

[0019] Preferably, the acid is acetic acid.

[0020] Preferably, the salt is sodium acetate.

[0021] Preferably, the molar ratio of compound 1 to compound 2 is 1:0.8 to 1.2, more preferably 1:1;

[0022] Preferably, the molar ratio of compound 1 to the catalyst is 100 to 120:1, more preferably 110:1;

[0023] Preferably, the molar volume ratio of compound 1 to the base is 1 mmol:3-5 mL, more preferably 1 mmol:4 mL;

[0024] Preferably, the molar ratio of compound 3 to compound 4 is 1:0.8 to 1.2, more preferably 1:1;

[0025] Preferably, the molar volume ratio of compound 3 to the acid is 1 mmol:0.5-3 mL, more preferably 1 mmol:1 mL;

[0026] Preferably, the molar ratio of compound 3 to the salt is 1:4 to 6, more preferably 1:4.

[0027] Preferably, the temperature of the two-step reaction is independently 20-140° C., and may be 100° C.; the time of the two-step reaction is independently 2 h to 12 h, and may be 12 h.

[0028] Preferably, in the reaction of compound 3 and compound 4, the organic solvent is one of N,N-dimethylformamide, toluene and acetonitrile, preferably N,N-dimethylformamide.

[0029] Preferably, all reactions are carried out under an inert atmosphere.

[0030] The third aspect of the present invention provides a fluorescent probe, which is a conjugate of the fluorescent dye described above and at least one of the following compounds: a drug, a protein or a peptide.

[0031] The fluorescent dye and fluorescent probe provided by the present invention can be used for fluorescence imaging, specifically imaging in the second near-infrared region. The fluorescent dye can also be used for imaging after conjugation modification of compounds such as drugs.

[0032] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0033] 1) Fluorescent dyes have carboxylic acid conjugated modification sites that can be further modified with drugs, proteins, etc.

[0034] 2) Fluorescent dyes and probes have good chemical stability and can be used for long-term in vivo imaging.

[0035] 3) The emission wavelength of fluorescent dyes and probes reaches the near-infrared region II, which can effectively reduce the impact of autofluorescence background signals in vivo;

[0036] 4) After injection into the tail vein, the fluorescent dye and probe can obviously light up the liver and then the intestine, indicating that the fluorescent dye and probe can be excreted from the body through the hepatobiliary metabolic pathway.

[0037] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present application. Moreover, the same reference numerals are used throughout the drawings to represent the same components. In the drawings:

[0039] Figure 1 The absorption and emission spectra of the fluorescent dye VIPI prepared in the present invention in chloroform, the horizontal axis is the wavelength, the vertical axis is the absorption or fluorescence intensity, and the concentration of the fluorescent dye is 10 μM; the left side is the absorption spectrum, and the right side is the emission spectrum.

[0040] Figure 2 This is a fluorescence imaging diagram of the fluorescent dye VIPI prepared in the present invention in mice. DETAILED DESCRIPTION

[0041] The following embodiments of the technical solution of the present application will be described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present application, and are therefore only used as examples, and cannot be used to limit the scope of protection of the present application.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which this application belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" in the specification and claims of this application and the above-mentioned figure descriptions and any variations thereof are intended to cover non-exclusive inclusions.

[0043] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0044] Example 1, preparation method of VIPI

[0045] Its synthetic route is as follows:

[0046]

[0047] Under N2 protection, a mixture of compound 1 (2mmol), compound 2 (2mmol), triethylamine (4mL) and Pd(OAc)2 (0.018mmol) was placed in DMF (20mL) and heated to reflux overnight at 100°C. After the reaction was completed, it was extracted with dichloromethane (100mL) and brine (100mL×3). Then, the organic layer was concentrated in vacuum and purified by column chromatography with silica gel (dichloromethane as eluent). An orange solid (yield 40%) was obtained as compound 3. Under N2 protection, compound 3 (0.4mmol), compound 4 (0.4mmol) and sodium acetate (4mmol) were refluxed in acetic acid (10mL) for 12h. After the reaction was completed, the mixture was concentrated in vacuum to form a blue-green solid, which was purified by column chromatography with silica gel (dichloromethane: methanol = 8:1). The fluorescent dye VIPI was obtained (the yield of this step was 40%). The prepared fluorescent dye VIPI was used in Example 3.

[0048] The structure of the fluorescent dye VIPI is confirmed as follows:

[0049] 1H NMR (400MHz, 298K, CD2Cl2) δ (ppm): 8.78 (d, J = 14.8Hz, 1H), 7.57 (t, J = 7.4Hz, 2H), 7 .49–7.42(m,5H),7.30(s,1H),7.21(d,J=16.0Hz,1H),7.07(s,2H),6.93(d,J=16.1H z,1H),6.42(d,J=14.8Hz,1H),4.29(t,J=7.4Hz,2H),3.27(t,J=5.6Hz,4H),2.85–2. 76(m,9H),2.54(t,J=7.0Hz,2H),2.03–1.97(m,8H),1.88(s,6H),1.66–1.59(m,3H).

[0050] 13 C NMR(100MHz,298K,CD2Cl2)δ(ppm):176.9,174.4,162.2,153.7,145.8,1 43.9,143.4,141.8,141.5,134.0,133.4,129.2,129.2,127.7,127.1,126 .1,123.6,123.2,122.5,121.4,120.5,120.5,115.5,112.4,111.7,103. 3,50.6,49.9,34.3,29.3,28.1,27.8,27.1,26.2,24.4,24.3,21.8,20.4.

[0051] High resolution mass spectrometry: C 45 H 49 N2O3 + , [M] + , the calculated value is 665.3738; the measured value is 665.3734.

[0052] Example 2: Absorption and emission spectra of VIPI in chloroform

[0053] Add the fluorescent dye VIPI to the test tube, and then add an appropriate volume of chloroform solution to make the final fluorophore concentration 10μM. Measure the absorption spectrum of the solution on a UV spectrophotometer, and measure its fluorescence spectrum on a fluorescence spectrophotometer. Select 720nm as the excitation wavelength to obtain the UV absorption and fluorescence emission spectra (see Figure 1 ).Depend on Figure 1 It can be seen that the fluorescent dye VIPI can emit strong fluorescence in an organic environment, and its maximum emission wavelength is 970nm.

[0054] Example 3. Near-infrared second window fluorescence imaging of fluorescent dye VIPI in mice.

[0055] 200 μL of fluorescent dye VIPI liposomes were injected into the mouse through the tail vein at an injection concentration of 500 μM. The whole body of the mouse was irradiated with an 808 nm laser and a 1000 nm long-pass filter. Clear and high-resolution images were observed (see Figure 2 ), and are mainly distributed in the liver and intestinal areas, indicating that the fluorescent dye VIPI can be well used in in vivo near-infrared second window imaging and has a clear hepatobiliary metabolic pathway.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them; although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application, and they should all be included in the scope of the claims and specification of the present application. In particular, as long as there is no structural conflict, the various technical features mentioned in the various embodiments can be combined in any way. The present application is not limited to the specific embodiments disclosed herein, but includes all technical solutions that fall within the scope of the claims.

Claims

1. A fluorescent dye in the second near-infrared region, the general structural formula of which is as follows: in, X is any one of F, Cl, Br and I.

2. The near-infrared second region fluorescent dye according to claim 1, characterized in that: X is Br.

3. A method for preparing the near-infrared second-region fluorescent dye according to claim 1, characterized in that: The synthetic route is as follows: Wherein, Y is any one of F, Cl, Br, and I, The base is any one of triethanolamine and triethylamine; The catalyst is palladium acetate; The acid is acetic acid; The salt is sodium acetate.

4. The method according to claim 3, characterized in that The substituent Y in the compound 1 is Br.

5. The method according to claim 3, characterized in that: The base is triethylamine.

6. The method according to any one of claims 3 to 5, characterized in that: The molar ratio of the compound 1 to the compound 2 is 1:0.8-1.

2.

7. The method according to any one of claims 3 to 5, characterized in that: The molar ratio of the compound 1 to the catalyst is 100-120:

1.

8. The method according to any one of claims 3 to 5, characterized in that: The molar volume ratio of the compound 1 to the base is 1 mmol:3-5 mL.

9. The method according to any one of claims 3 to 5, characterized in that: The molar ratio of the compound 3 to the compound 4 is 1:0.8-1.

2.

10. The method according to any one of claims 3 to 5, characterized in that: The molar volume ratio of the compound 3 to the acid is 1 mmol:0.5-3 mL.

11. The method according to any one of claims 3 to 5, characterized in that: The molar ratio of the compound 3 to the salt is 1:4-6.

12. The method according to any one of claims 3 to 5, characterized in that: The temperature of the two-step reaction is independently 20 to 140° C.; the time of the two-step reaction is independently 2 h to 12 h.

13. The method according to claim 12, characterized in that The temperature of the two-step reaction was 100°C; The two-step reaction time is 12 h.

14. The method according to any one of claims 3 to 5, characterized in that: The organic solvent in the reaction between the compound 3 and the compound 4 is any one of N,N-dimethylformamide, toluene and acetonitrile.

15. The method according to claim 14, characterized in that In the reaction between compound 3 and compound 4, the organic solvent is N,N-dimethylformamide.

16. The method according to any one of claims 3 to 5, characterized in that: All reactions were carried out under an inert atmosphere.

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